Synaptogenic protein tagged with biotin and reconstitution of  artificial synapse by using same

ABSTRACT

Disclosed is a polypeptide containing an extracellular domain of a synaptogenic protein, and a method for manufacturing a nerve cell, a complex containing a biotin tagged at the C-terminus of the polypeptide, an artificial synapse inducer for coupling the composite to a streptavidin (SAV)-coated substrate and a nerve cell. The complex tagged with a biotin at the C-terminus of the polypeptide containing the extracellular domain of the synaptogenic protein, such as neuroligin-1, can display activity by being attached to the SAV-coated substrate to adjust the orientation thereof without help of a supported lipid bilayer. The complex containing an additional RFP between the extracellular domain and the biotin of the synaptogenic protein not only facilitates easier mass-production, quantification, and tracking, but also displays activity of a normal synaptogenic protein, thereby inducing excitatory or inhibitory synaptic differentiation by being fixed to the substrate and added to the nerve cell culture.

TECHNICAL FIELD

The present invention relates to a complex comprising a polypeptidecomprising an extracellular domain of a synaptogenic protein; and biotintagged at the C-terminus thereof; an artificial synapse inducer, inwhich the above complex is attached to a substrate coated withstreptavidin (SAV); and a method for preparing a presynapticdifferentiation-induced neuron comprising culturing a neuron in a mediumcomprising the artificial synapse inducer.

BACKGROUND ART

Neurons convey information electrically and chemically in ahighly-organized system. Electrical impulses, known as action potentials(“AP”, hereinafter), traveling along axons converge at presynapticterminals and are converted into chemical signals, which areneurotransmitters. However, not all the transmitted APs triggerneurotransmitter release at the synaptic junctions, and not all thereleased neurotransmitters effectively induce postsynaptic APs. Unlikeexcitatory synapses, at the inhibitory synapses, APs are sequestered forthe purpose of orchestration of overall network communication.Therefore, it is essential to distinguish between the excitatory andinhibitory signals in order to understand, mimic, and monitor neuralnetwork behavior.

In biological systems, excitatory and inhibitory synapses are determinedby synaptic cell adhesion molecules (“CAMs”, hereinafter). Among theinteractions between the synaptic CAMs, the trans-synaptic adhesionbetween postsynaptic neuroligins (“Nlgs”, hereinafter) and presynapticneurexins (“Nrxs”, hereinafter) is most representative and has been mostextensively studied. Scheiffele et al. showed that the Nlgs expressed innon-neuronal cells were sufficient to induce presynaptic differentiationby introduction of presynaptic Nrxs. Furthermore, purified Nlg-1, whosetransmembrane domain (TMD) is swapped with glycosylphosphatidylinositol(GPI)-anchoring motif, can successfully induce presynapticdifferentiation when docked on glass microbeads that were coated withsupported lipid bilayer (SLB) membranes. However, the chemicalconjugation of Nlg-1 on polystyrene beads, despite its capability ofadhering to Nrx-expressing cells, failed to induce presynapticdifferentiation, suggesting that Nlg-1 requires a fluidic lipid bilayerenvironment for its activity. Additionally, the Nlg-1 is known to form adimer.

DISCLOSURE Technical Problem

The present inventors have studied a method for establishing anexcitatory artificial synapse via an orientation-controlledimmobilization of a synaptogenic protein on a substrate without a lipidbilayer. As a result, they have discovered that a synaptogenic protein,e.g., a protein complex in which biotin is tagged at the C-terminus ofNlg-1, can be immobilized on a substrate in a constant orientation by aSAV-biotin conjugation without the help of a lipid bilayer, and thethus-immobilized Nlg-1 can maintain its activity while being present inthe form of a dimer as is in vivo, and thus can form an artificialsynapse by inducing an excitatory presynaptic differentiation, therebycompleting the present invention.

Technical Solution

An object of the present invention is to provide a complex comprising apolypeptide comprising an extracellular domain of a synaptogenicprotein; and biotin tagged at the C-terminus of the polypeptide.

Another object of the present invention is to provide an artificialsynapse inducer, in which the above complex is attached to a substratecoated with SAV.

A further object of the present invention is to provide a method forpreparing a presynaptic differentiation-induced neuron comprisingculturing a neuron in a medium comprising the artificial synapseinducer.

Advantageous Effects of the Invention

The complex of the present invention, which is tagged with biotin at theC-terminus of a polypeptide containing an extracellular domain of asynaptogenic protein, such as Nlg-1, can exhibit its activity by beingattached to a substrate coated with SAV, thereby enabling the control ofits orientation without the help of a lipid bilayer. Additionally, whenthe complex further includes RFP between the extracellular domain of asynaptogenic protein and biotin, it can not only facilitate itsmass-production, quantification, and tracking but also exhibit theactivity of a normal synaptogenic protein. Accordingly, the complex caninduce an effective and excitatory or inhibitory synapse differentiationby addition thereof into a nerve cell culture after being immobilized ona substrate, and thus can be used as an artificial synapse inducer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a shows a 3-dimensional crystal structure of a neuroligin-1(Nlg-1) dimer and neurexins (Nrx) complex. As shown on the left, Nrx isplanted on the presynaptic membrane while Nlg-1 on the postsynapticmembrane. On the right, the structure of the two central α-helicesrequired for Nlg-1 dimerization is shown.

FIG. 1b shows a schematic diagram of a plasmid constitution for thepreparation of an Nlg-1 complex used in the present invention, whichcontains a fluorescence protein and/or biotin. In the figure, HAindicates HA tag; H6, hexa-His tag; OG, O-glycosylation-rich domain;TMD, transmembrane domain; CD, cytoplasmic domain; GPI, GPI-anchoringmotif; GS, glycine-serine linker; and AP, biotin acceptor peptide orAviTag.

FIG. 2 shows pictures illustrating western blot profiles for thepurification of an Nlg-1 complex to which a fluorescent protein, etc.,is conjugated. YFP-Nlg-1-GPI, which is Nlg-1 tagged with YFP at theN-terminus and tagged with GPI at the C-terminus, rarely appeared in theelution fraction despite its presence in total cell lysate. When the GPImotif was replaced with AP, the YFP-Nlg-1-AP was secreted into theculture medium and retained in Ni-NTA resin. Both in vitro and in vivobiotinylation using BirA enzyme revealed biotinylated Nlg-1, regardlessof the position of fluorescence proteins, YFP and RFP. An asteriskcorresponds to the size of the Nlg-1 complex. A dagger corresponds tothe non-specific binding of SAV-HRP to albumin in the culture medium. Adouble dagger indicates externally added BirA enzyme. RN, RFP-Nlg-1-AP;NR, Nlg-1-RFP-AP.

FIG. 3 shows a picture illustrating the quantification of Nlg-1-RFP-APusing electrophoresis and western blot. A known amount of BSA was loadedand compared with a known volume of an Nlg-1-RFP-AP elution fraction toyield Nlg-1 concentration (left panel). The biotinylated band wasverified using western blot (right panel). The correlation between thefluorescence intensity and the concentration of Nlg-1-RFP-AP was alsoexhibited.

FIG. 4 shows pictures illustrating the reconstitution of fluorescent andbiotinylated Nlg-1 on a biotin-functionalized SLB membrane and DynabeadsSAV. YN indicates YFP-Nlg1-AP; RN, RFP-Nlg-1-AP; NR, Nlg-1-RFP-AP. Scalebar=100 μm.

FIG. 5 shows pictures illustrating that the N-terminally tagged YFP inNlg-1 on SLB beads (a) and Dynabeads (b) exerted negligiblefunctionality in terms of synapsin I and VGlut1 aggregation. ReplacingYFP with photostable, monomeric TagRFP-T slightly improved the Nlg-1activity ((c) and (d), arrows). Scale bar=10 μm.

FIG. 6 shows pictures illustrating the presynaptic differentiationinduced on artificial substrates containing beads coated with Nlg-1complex according to the present invention. SLB beads coated with Nlg-1complex and Dynabeads coated with SAV were used as experimental groups,and PDK beads (poly-D-lysine beads) were used as a comparative group.Nlg-1-RFP-AP (NR) immobilized on SLB beads (a) and Dynabeads SAV (b),and PDK-coated beads (c) recruited synapsin I and VGlut1 and assembledaround the beads. (d) shows a graph illustrating the ratio of meanfluorescence intensity (MFI) between VGlut1 and synapsin I measured inthe same region of interest (ROI) around the beads.

FIG. 7 shows pictures illustrating the presynaptic differentiationinduced on artificial substrates containing beads coated with an Nlg-1complex according to the present invention. SLB beads coated with theNlg-1 complex and Dynabeads SAV were used as experimental groups, andPDK beads were used as a comparative group. (a) to (c) show picturesrespectively illustrating the expression of synapsin I and Bassoon ineach experimental group and control group, and they show that theenhanced synapsin I immunofluorescence puncta always colocalize withBassoon-positive puncta.

FIG. 8 shows pictures illustrating the presynaptic differentiationinduced on artificial substrates containing beads coated with an Nlg-1complex according to the present invention. SLB beads coated with theNlg-1 complex and Dynabeads SAV were used as experimental groups, andPDK beads were used as a comparative group. (a) to (c) show picturesrespectively illustrating the comparison between excitatory andinhibitory presynaptic differentiations induced by NR SLB beads (a), NRDynabeads SAV (b), and PDK beads (c). VGlut1 and GAD were used asmarkers for excitatory and inhibitory presynaptic differentiations,respectively. (d) shows a graph illustrating the ratio of meanfluorescence intensity (MFI) between GAD and VGlut1-positive punctaaround the beads. The diameter of silica beads and Dynabeads was 5 μmand 2.8 μm, respectively.

FIG. 9 shows pictures illustrating the abilities for distinctlysurface-treated beads to induce synapsin I and VGlut1. A complexC-terminally tagged with FRP regenerated Nla-1 activity both on SLB (b)and non-SLB membranes (c). PDK beads showed less frequent synapsin I-and VGlut1-positive fluorescence puncta (d). (a) shows the result frombiotinylated BSA-coated beads as a negative control (scale bar=10 μm).

FIG. 10 shows pictures illustrating that the selected NR SLB beads (a)and NR Dynabeads (b) show synapsin I- and VGlut1-positive puncta. (c)PDK beads show weaker VGlut1-positive fluorescence puncta.

FIG. 11 shows pictures illustrating the verification of aggregatedsynapsin I aggregation as a positive presynapse marker. (a) shows apicture illustrating the measurement result of inactive biotinylated BSAon SLB beads, and (b) and (c) show the induction of both synapsin I andBassoon due to NR, with the latter being nearer to the beads. (d) showsa picture illustrating the aggregation of the marker proteins by PDKbeads, in which PDK beads show less frequent aggregation of the markerproteins. Scale bar=10 μm.

FIG. 12 shows pictures illustrating the preference for excitatorypresynaptic differentiation of NR. (a) shows a picture illustrating themeasurement result of SLB beads coated with inactive biotinylated BSA,as a negative control, showing background GAD puncta. (b) and (c) showthe selectivity on excitatory presynaptic differentiation due to NR, inwhich NR induced VGlut1 but not GAD. (d) shows the measurement result ofPDK beads, in which bead-shaped GAD-positive only puncta (arrow) andbead-shaped mixed puncta (arrowhead) were observed. Scale bar=10 μm.

FIG. 13 shows a schematic diagram illustrating a presynapticdifferentiation mechanism induced by the Nlg-1 complex of the presentinvention and polybasic molecules. N-terminally tagged RFP (inside thedotted circles) may keep Nlg-1 (grey) from binding with Nrx (lightgrey), resulting in poor synaptogenesis at contacting neurites (left).An Nlg-1 complex with a C-terminal modification with RFP, in which theintroduced RFP does not affect the interaction between an Nlg-1 dimerand Nrxs, can induce a successful excitatory presynaptic differentiation(middle). Additionally, both of the above two complexes can interactwith Nrx of the contacting neurites through biotin conjugated to theC-terminus, without the supported lipid bilayer (SLB) membrane, by beingimmobilized on the artificial substrate coated with SAV in a uniformorientation. A differentiation-inducing mechanism by polybasic moleculessuch as PDK and PE is shown, and these materials can also inducepresynaptic differentiation via the interaction with proteoglycans innegative charges, but they do not show any defined preference forexcitatory synapses by an Nlg-1 complex and also have a lowerdifferentiation-inducing efficiency (right).

FIG. 14 shows pictures illustrating the difference between the synapsesbeing induced by Nlg-1 beads and PDK beads, which were added duringdifferent stages of neuron development, respectively. The pictures showfluorescence images of immunocytochemistry regarding synapse I ofneurons differentiated for 18 days in vitro (18 DIV). (a) to (e) showneurons which were treated with Nlg-1 beads and PDK beads in cultivationat the 0 DIV, 7 DIV, 10 DIV, 14 DIV, and 17 DIV in vitro, respectively,and continuously developed until the 18 DIV, i.e., (a) to (e) representneurons cultured by allowing them to come into contact with the beadsadded thereto for 18 days, 11 days, 8 days, 4 days, and 1 day. The beadsindicated with an arrowhead with DIC images (left) are Nlg-1 beads, andthe other beads are PDK beads.

EMBODIMENTS

In an aspect to accomplish the above objects, the present inventionprovides a complex comprising a polypeptide comprising an extracellulardomain of a synaptogenic protein; and biotin tagged at the C-terminus ofthe polypeptide.

As used herein, the term “synaptogenic protein” collectively refers toall the proteins that can mediate the synaptogenesis by inducing theinitial contact between an axon and a target cell thereof. The termsynaptogenic protein, which may also be called synapse-forming protein,will be used in the present invention. The synaptogenic protein isinvolved in the introduction and organization of presynaptic andpostsynaptic proteins required for synaptic transmission. For example,the synaptogenic proteins such as neuroligin, leucine-rich repeattransmembrane protein (LRRTM), netrin G ligand (NGL), synaptic celladhesion molecule (SynCAM), ephrin-B receptor (EphB), and Slit- andTrk-like proteins (Slitrk), which are present postsynaptically, caninduce presynaptic differentiation. Meanwhile, synaptogenic proteinssuch as neurexin, leukocyte common antigen-related protein (LAR), andnetrin G, which are present presynaptically, can induce postsynapticdifferentiation. Preferably, the synaptogenic protein may be Nlg, Nrx,LRRTM, NGL, SynCAM, EphB, LAR, netrin G, or Slitrk, and more preferablyNlg-1, but is not limited thereto. The synaptogenic proteins commonlyinclude a target-binding site in the N-terminal direction while having atransmembrane domain in the C-terminal direction. That is, theN-terminus includes an extracellular domain which exhibits an activityand the C-terminus includes a transmembrane domain which is attached toa cell membrane.

The present invention is characterized in that the synaptogenic proteincan be fixed to a substrate by the SAV-biotin binding while maintainingthe intrinsic activity of the synaptogenic protein by conjugating biotinto the C-terminus of the extracellular domain of the synaptogenicprotein. This complex can induce an excitatory or inhibitory synapticdifferentiation when it is used in cultivation of neuron in the formbeing fixed to SAV, which is coated on the substrate via biotinconjugated to the C-terminus. Additionally, this complex does notrequire a lipid bilayer, which was conventionally introduced for thecontrolled orientation of the synaptogenic protein, and it can alsoremove a transmembrane domain, etc., as long as it includes anextracellular domain capable of exhibiting the activity of thesynaptogenic protein.

For example, the complex according to the present invention ischaracterized in that it includes an extracellular domain of Nlg-1,which is a protein mediating the formation of a synapse between neurons,and its C-terminus is conjugated to biotin, and it is thus capable ofbeing fixed on the artificial substrate coated with SAV via the biotinconjugated to the C-terminus to thereby have a controlled orientation.

As used herein, the term “neuroligin-1 (Nlg-1)” refers to a type Imembrane protein which is present in the postsynaptic membrane andmediates the formation of a synapse between neurons. Nlg mediatessignaling through synapses and affects the properties of a neuralnetwork by specifying synaptic functions. An alteration in genesencoding Nlgs in humans may result in autism and other cognitivedisorders. This suggests that the expression of Nlg can inducepresynaptic differentiation, which is mediated by the contact, at axonscontacted thereto. The extracellular domain of Nlg mostly consists of aregion that is homologous to acetylcholinesterase (AChE), and the aminoacids important for the catalysis in AChE are not conserved in Nlg, andthus Nlg lacks esterase activity. Additionally, the AChE homologousregion is crucial for the proper function of Nlg. Nlgs act as ligandsfor β-Nrxs which are located presynaptically. Nlg and β-Nrx “shakehands”, resulting in the connection between two neurons and theproduction of a synapse. Nlgs also act in honeybees and their functionsin insects are similar to those of vertebrates. Nlg dysfunction has beenimplicated in autistic spectrum disorders.

As used herein, the term “neurexin (Nrx)” refers to a presynapticprotein that helps to connect neurons at the synapse. Nrx is a type Imembrane protein and classified into two kinds, α-Nrx and β-Nrx. Theα-Nrxs are the larger of the two and have a different amino-terminalextracellular sequence. Nrx mediates signaling across synapses, andaffects the properties of a neural network by specifying the synapticfunctions. An alteration in genes encoding neurexin in humans may induceautism and other cognitive disorders. The β-Nrxs located presynapticallyact as receptors for neuroligins located postsynaptically. As describedabove, Nlgs and β-Nrx “shake hands”, resulting in the connection betweentwo neurons and the production of a synapse. Additionally, β-Nrxs areinvolved in angiogenesis.

As used herein, the term “leucine-rich repeat transmembrane protein(LRRTM)” refers to a protein which recognizes the protein labelingpresent on the surfaces of other neurons. Numerous cells canspecifically form synapses with the constituting components atparticular subcellular levels of target cells. During this process, theLRRTM can recognize particular proteins, similar to a specificantigen-antibody recognition in an immune response, thereby enabling therecognition of target cells.

As used herein, the term “netrin G” refers to a protein immobilized onan axonal membrane by glycosylphosphatidylinositol (GPI), andvertebrates have its isoforms, netrin-G1 and netrin-G2.

As used herein, the term “netrin G ligand (NGL)” refers to a ligandwhich specifically binds to netrin G, and it is located on thepostsynaptic membrane and interacts with presynaptic netrin G. Netrin Gand NGL thereof serve as a modulatory signaling system to synapses, andthus any deficiency thereof may lead to behavioral defects.

As used herein, the term “synaptic cell adhesion molecule (SynCAM)”refers to a hemophilic protein which includes the transmembraneIg-domain, also known as TSLC1, Sg1GSF, or IGSF4, and includes anintracellular PDZ protein-binding motif. Originally, it was identifiedas a tumor-inhibiting factor against small lung cell carcinoma, but itis now known to be mainly involved in intracellular adhesion andformation of synapses. Most SynCAMs are located on the synapses wherethe assembly and differentiation of synapses start through the centralnervous system.

As used herein, the term “ephrin-B receptor (EphB)” refers to a receptorprotein which interacts with ephrin-B, i.e., a ligand family thereof,and is activated by binding to ephrin-B. The ephrin-B is a subfamily ofthe Eph tyrosine kinase receptor family, which is composed of sixmembers of EphB1 to EphB6. The extracellular domain of ephrin receptorsincludes a cysteine-rich region, two fibronectin type III domains, and ahighly conserved globular ephrin ligand-binding domain, and thecytoplasmic domain includes two conserved tyrosine residues, a tyrosinekinase domain, a sterile alpha motif (SAM), and a juxtamembrane regionhaving a PDZ-binding motif.

As used herein, the term “Slit- and Trk-like proteins (Slitrk)” refersto a neural transmembrane protein, which regulates the growth ofneuritis, and it s a kind of a synapse organizer. The Slitrks areabundantly present in postsynaptic densities and their overexpressionpromotes the formation of synapses. The Slitrks are known to be involvedin the formation of both excitatory and inhibitory synapses in anisoform-dependent manner. Additionally, Slitrks, along with leukocyteantigen-related receptor protein tyrosine phosphatase (LAR-RPTP) familymembers, maintain the formation of synapses so that theexcitatory-inhibitory balance can be harmonized.

As used herein, the term “biotin” refers to a water-soluble vitamin B(vitamin B₇), which is also called vitamin H or coenzyme R. Biotinconsists of a ureido (tetrahydroimidizalone) ring fused with atetrahydrothiophene ring. Biotin contains valeric acid bound to a carbonatom of the tetrahydrothiophene ring. Biotin is a cofactor forcarboxylase and is involved in the syntheses of fatty acids, isoleucine,and valine, and gluconeogenesis. Biotin, in addition to itscharacteristic as a cofactor described above, has the characteristics ofstrongly binding to proteins, such as avidin, SAV, and neutravidin (ordeglycosylated avidin), with a dissociation constant K_(d) at the levelof 10⁻¹⁴ M to 10⁻¹⁵ M. In particular, since the specific binding ofbiotin with SAV can be maintained in harsh conditions, the SAV-biotinbinding has been applied in various bioengineering fields. Due to thesmall size of biotin, it does not affect on the activities of proteinscomprising the same and thus biotin is attached to various proteins tobe used in biochemical assays, etc. This process, i.e., the process ofattaching biotin to proteins, is called biotinylation. The biotinylatedproteins can be immobilized on the beads by incubating the biotinylatedproteins with SAV/avidin beads, etc.

As described above, the complex according to the present inventioncontains biotin attached to its C-terminus. The attachment of biotin tothe C-terminus may be performed using any method known in the artwithout limitation. For example, the above complex may be prepared invivo production and secretion in a state where biotin is attached to theC-terminus of the complex, or via in vitro biotinylation after in vivoproduction and secretion in a state where a biotin acceptor peptide,e.g., AP or AviTag, is tagged.

Preferably, the complex according to the present invention may comprisean extracellular domain of Nlg-1 having an amino acid sequencerepresented by SEQ ID NO: 1. However, the amino acid sequence is notlimited by the native amino acid sequence of SEQ ID NO: 1, but anymutein of the native amino acid sequence or a fragment thereof may beincluded, as long as they can exhibit an activity as Nlg-1, for example,as long as they can induce synaptic differentiation. As used herein, theterm “mutein” refers to a protein with an amino acid sequence altered bydeletion, addition, non-conservative or conservative substitution, or acombination thereof in at least one amino acid residue of a given nativeamino acid sequence. It is obvious that this can be equally applied tosynaptogenic proteins other than Nlg-1.

The complex according to the present invention may further comprise afluorescent protein, and the fluorescent protein may be comprised in thesequential order of a polypeptide comprising an extracellular domain ofa synaptogenic protein, a fluorescent protein, and biotin from itsN-terminus. The use of the fluorescent protein enables purification,quantification, and tracking of the complex according to the presentinvention.

As used herein, the term “fluorescent protein” refers to a member of aclass structurally corresponding to proteins which commonly shareself-sufficient intrinsic characteristics and are capable of formingchromophores with wavelengths of visible light from three amino acidsequences within their polypeptide sequences. Localization of a geneproduct and dynamics can be visualized using a fluorescence microscopeby introducing a gene (or a chimeric gene) into a live cell and allowingit to encode an engineered fluorescent protein. Accordingly, thefluorescent proteins are effectively used in numerous bioengineeringstudies. As described above, fluorescent proteins are most frequentlyused in research for imaging of the localization and dynamics ofparticular organelles or recombinant proteins in a live cell. For thevisualization of particular organelles, a gene encoding a fluorescentprotein may be fused to a cDNA encoding a protein or peptide which isknown to be localized in particular organelles to be observed, using astandard molecular biology technique. The fusion can be performed byforming a covalent link between a target motif and a fluorescent proteinand expressing the chimeric gene as a single polypeptide. A mammaliancell may be transfected with a chimeric gene-containing plasmid under anappropriate promoter in order to express the chimeric gene and producethe corresponding protein. The chimera is set to be located on thetarget organelles so that the target organelles can emit fluorescence.Accordingly, the shapes, dynamics, and distribution of organelles can berepresented as a function of time using a fluorescence microscope.Additionally, information on multiple organelles can be obtainedsimultaneously using fluorescent proteins of various colors.

Preferably, the fluorescent protein may be blue fluorescent protein(BFP), enhanced blue fluorescent protein (eBFP), cyan fluorescentprotein (CFP), enhanced cyan fluorescent protein (eCFP), greenfluorescent protein (GFP), enhanced green fluorescent protein (eGFP),yellow fluorescent protein (YFP), enhanced yellow fluorescent protein(eYFP), or red fluorescent protein (RFP). Some of these fluorescentproteins require a high energy source close to that of UV rays for theexcitation. However, high energy light has low transmittance. Therefore,when the light is applied in cells, it may cause harmful effects such asgenetic mutations, or it may cause adverse effects on cells through theproducts generated by photoreactions. Meanwhile, some fluorescentproteins may tend to show multimerization of dimers or more. However,for some fluorescent proteins with a high tendency to multimerize, themultimerization may affect the orientation or activities of synaptogenicproteins, and thus it is preferable to use fluorescent proteins with alow tendency to multimerize. Accordingly, the fluorescent protein may beYFP or RFP, but is not limited thereto, and most preferably, thefluorescent protein may be RFP.

The complex according to the present invention may further comprise apolyhistidine-tag (His-tag) or an influenza hemagglutinin epitope tag(HA-tag) for its separation and purification.

According to a specific embodiment of the present invention, when afluorescent protein is located in the N-terminal direction of Nlg-1, itmay inhibit Nlg-1 from forming a synapse by blocking binding Nlg-1 toNrx. However, when the fluorescent protein was introduced into theC-terminal direction, it was confirmed that the differentiation ofneurons could be normally induced by Nlg-1 (FIG. 13).

In another aspect, the present invention provides an artificial synapseinducer in which the above complex is attached to a substrate coatedwith a biotin-binding protein.

As used herein, the term “biotin-binding protein” refers to a proteinwhich can specifically bind to biotin with high binding affinity. Sincethe biotin-binding protein has high specificity to biotin, it has a lowlevel of non-specific binding. Regarding the binding to biotin, thebiotin-binding has a dissociation constant of 10⁻¹⁴ M to 10⁻¹⁵ M, andthus it can maintain the binding under very harsh conditions. Thebiotin-binding protein is a tetramer and each protein molecule canmaximally bind to four biotin molecules.

Preferably, the biotin-binding protein may be an avidin-like proteinssuch as SAV, traptavidin, or neutravidin, but any protein may be usedwithout limitation, as long as it can specifically bind to biotin.

As used herein, the term “avidin” refers to a biotin-binding proteinwhich is considered to act as an antibiotic in eggs of birds, reptiles,and amphibians. In the case of chicken avidin, it has a molecular weightof 67 kDa to 68 kDa, is formed from four small units consisting of 128amino acids, respectively, and each small unit can bind to a singlebiotin molecule. Since the avidin is highly glycosylated, it containscarbohydrates in the amount of 10% of the total mass, has a basicisoelectric point (pI) from 10 to 10.5, and has high solubility in waterand aqueous salt solutions.

As used herein, the term “SAV” refers to a tetramer biotin-bindingprotein with a molecular weight of 60 kDa, which is separated fromStreptomyces avidinii. SAV has a very low homology with avidin, buttheir structures are very similar to each other. SAV has an antibioticactivity, as is the case with avidin, and has very high binding affinityto biotin. Meanwhile, unlike avidin, SAV does not contain carbohydrates,has an acidic isoelectric point (pI=5), and has a significantly lowersolubility compared to that of avidin. Commercially available SAV, e.g.,Thermo Scientific Pierce SAV, is SAV in a recombinant form with amolecular weight of 53 kDa having an isoelectric point close to neutral(pI=6.8 to 7.5). The lack of glycosylation and low pI of SAV result in alow level of non-specific binding (in particular, lectin binding)compared to that of avidin. Due to these characteristics, SAV isselected as an ideal reagent for many detection systems.

As used herein, the term “traptavidin” refers to a variant or mutein ofSAV which has an about 10 times slower dissociation rate to biotin,increased mechanical strength, and improved thermal stability.Traptavidin also binds specifically to biotin.

As used herein, the term “neutravidin”, also called deglycosylatedavidin, refers to a protein prepared for the purpose of resolving themajor drawbacks of native avidin and SAV. As presented in its name, itis a protein prepared by the deglycosylation of avidin, and whichmaintains high binding affinity to biotin while having a reducedmolecular weight (60 kDa) compared to that of avidin. Thedeglycosylation of avidin reduces the lectin binding to an undetectablelevel and lowers the isoelectric point (pI=6.3), thereby effectivelyremoving the major causes of non-specific binding to avidin. Sincelysine residues are maintained in a usable state, neutravidin can beeasily derivatized or complexed, as is the case with SAV. Additionally,since neutravidin exhibits high binding affinity to biotin and a lownon-specific binding, it can be used variously as an idealbiotin-binding protein.

As used herein, the term “substrate” may refer to a material in solidphase with a predetermined shape, which can support the complexaccording to the present invention to be immobilized thereto. Thematerials to be used as the substrate may include silicone, glass,metals, magnetic materials, semi-conductors, ceramics, etc., withoutlimitation. Additionally, the substrate may be modified on its surfaceto have reactivity, or further introduced with a layer of a newmaterial. The shape of the substrate may be in various forms such as asphere, plane, etc., but is not limited thereto. Preferably, thesubstrate may be the shape of in spherical microbeads having a diameterat the level of micrometers.

As used herein, the term “artificial synapse inducer” refers to amaterial which can induce synaptic differentiation of neurons in an invitro condition, instead of an in vivo differentiation environment ofneurons.

According to a specific embodiment of the present invention, it wasconfirmed that by culturing artificial synapse inducers according to thepresent invention, in which the Nlg-1 complex comprising biotin at itsC-terminus was attached to microbeads along with neurons, synapsin I,which is a presynaptic marker protein, and vesicular glutamatetransporter 1 (VGlut1) were introduced around the inducers to inducepresynaptic differentiation (FIGS. 6a to 6d , and FIG. 9).

In another aspect, the present invention provides a method for preparinga presynaptic differentiation-induced neuron comprising culturing theneuron in a medium comprising the artificial synapse inducers.

Preferably, an excitatory presynaptic differentiation or an inhibitorypresynaptic differentiation can be induced using the artificial synapseinducers according to the present invention. The selectivity on thedifferentiation direction varies according to the kinds of synaptogenicproteins introduced in the artificial synapse inducers.

As used herein, the term “an excitatory synapse” refers to a synapse inwhich an action potential in a presynaptic neuron increases theprobability of occurrence of an action potential in a postsynaptic cell.Neurons form networks through which nerve impulses travel, each neuronmaking numerous connections with other neurons. These electrical signalsmay be excitatory or inhibitory, and, if the total of excitatoryinfluences exceeds that of the inhibitory influences, the neuron may bestimulated. That is, a new action potential may be generated at its axonhillock, thereby transmitting the information to yet another cell. Thisphenomenon is known as an excitatory postsynaptic potential (EPSP). Itmay occur via direct contact between cells (i.e., via gap junctions), asin an electrical synapse, however, it most commonly occurs via thevesicular release of neurotransmitters from the presynaptic axonterminal into the synaptic cleft, as in a chemical synapse. Theexcitatory neurotransmitters then migrate via diffusion to the dendriticspine of the postsynaptic neuron and bind a specific transmembranereceptor protein that triggers the depolarization of the cell.

Meanwhile, as used herein, the term “an inhibitory synapse” refers to asynapse in which a nerve impulse in a presynaptic cell induces therelease of inhibitory neurotransmitters that triggers the opening ofmultiple ion channels in the postsynaptic cell membrane so that negativeions move into (or positive ions move out of) the cell, therebystabilizing its resting potential. One representative example of theinhibitory neurotransmitter is GABA.

The excitatory synapses play an important role in information processingwithin the brain and throughout the peripheral nervous system. Generallylocated on the dendritic spines, or neuronal membrane protrusions onwhich glutamate receptors and postsynaptic density (PSD) components areconcentrated, the excitatory synapses aid in the electrical transmissionof neuronal signals. The physical morphology of synapses is crucial inunderstanding their functions, and the inappropriate loss of synapticstability leads to the disruption of neuronal circuits and thesubsequent neurological diseases. Despite the presence of innumerabledifferent causes for different neurodegenerative illnesses, such asgenetic dispositions or mutations, normal aging process, parasitic andviral causes, etc., many can be traced back to dysfunctional signalingbetween the neurons themselves, often at the synapse. Excitatorymechanisms are involved in various conditions leading to neuronaldamage, including hypoglycemia, trauma, stroke, seizures, and manyneurodegenerative diseases, thus having important implications indisease treatment. Therefore, there is a need for the study toindependently study the excitatory signaling and the inhibitorysignaling in order to understand, mimic, and observe the behaviors ofneuronal networks.

The selective synaptic differentiation trend according to the kinds ofmutually interacting presynaptic and postsynaptic proteins aresummarized in Table 1 below. For example, as shown in Table 1, anexcitatory synaptic differentiation can be selectively induced when eachof presynaptic β-Nrx, α-Nrx or β-Nrx (-SS4), netrin G-1 or netrin G-2,and LAR-RTPT interacts with postsynaptic Nlg-1, LRRTM 1 or LRRTM 2,NGL-1 or NGL-2, and NGL-3, respectively. Meanwhile, an inhibitorysynaptic differentiation can be selectively induced when presynaptic PTPand postsynaptic Slitrk-3 interact with each other, and the excitatorysynaptic differentiation and the inhibitory synaptic differentiation canbe induced simultaneously when each of presynaptic α-Nrx and PTPinteracts with postsynaptic Nlg-2 and Slitrk-1, -2, -4, -5, or -6,respectively. Accordingly, an artificial synapse inducer comprising anappropriate synaptogenic protein can be selected according to thedirection of the synaptic differentiation to be induced.

TABLE 1 Interactions between synapse-forming proteins SynapsePresynaptic Postsynaptic Excitatory Inhibitory β-Nrx Nlg-1 O α-Nrx Nlg-2O O α-, β-Nrx (-SS4^([1])) LRRTM 1 and O LRRTM 2 Netrin G-1, netrin G-2NGL-1 and NGL-2 O LAR-RPTP^([2]) NGL-3 O PTP^([3]) Slitrk-1, -2, -4, -5,O O and -6 PTP^([3]) Slitrk-3 O ^([1])-SS4: Lack of splice site 4(adhesion part 4) ^([2])LAR-RPTP: Leukocyte antigen-related receptorprotein tyrosine phosphatase ^([3])PTP: Protein tyrosine phosphatase

According to a specific embodiment of the present invention, whenneurons are cultured along with artificial synapse inducers according tothe present invention, in which the Nlg-1 complex comprising biotin atits C-terminus, immobilized to a lipid bilayer or the beads coated withSAV, synapsin I, which is a representative presynaptic marker, andVGlut1, which is an excitatory presynaptic marker protein, were shown tohave positive responses. Additionally, they also showed strong positiveresponses to Bassoon, which labels the presynaptic active zone.Meanwhile, it was confirmed that GAD, which is a marker protein for aninhibitory γ-aminobutyric acid synapse, was not expressed. In contrast,the PDK beads, which are conventionally known to induce a presynapticdifferentiation, were shown to significantly reduce the expression ofVGlut1, which is an excitatory presynaptic marker, compared to when theinducers according to the present invention were used, and also shown toexpress GAD, an inhibitory presynaptic marker (FIGS. 6 to 8, and FIG.10). This suggests that the artificial synapse inducers according to thepresent invention, which includes the Nlg-1 complex to which biotin isconjugated at its C-terminus, can be effectively used for inducing anexcitatory presynaptic differentiation.

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, the present invention will be described in more detail withreference to the following Examples. However, these Examples are forillustrative purposes only, and the invention is not intended to belimited by these Examples.

Example 1 Materials

A plasmid encoding cholinesterase-like domain (CLD) of Nlg-1 followed byGPI anchoring motif (pNICE-HA-H6-Nlg-1-GPI) was provided by Dr. PeterScheiffele. A plasmid carrying EYFP-tagged full length Nlg-1(pNICE-YFP-Nlg-1) was provided by Dr. Ann Marie Craig. A TagRFP-Texpressing plasmid (pcDNA3-TagRFP-T) was provided by Dr. Roger Y. Tsien.BirA plasmids (pDisplay-BirA-ER and pET21a-BirA) were purchased fromAddgene (Cambridge, Mass.). Carbenicillin (Carb) was purchased from GoldBiotechnology (St. Louis, Mo.). Streptavidin (SAV), bovine serum albumin(BSA) (A3059), adenosine 5′-triphosphate (ATP), polyethyleneimine (Mw:25,000), Kanamycin sulfate, and G418 (Geneticin) were purchased fromSigma-Aldrich (St. Louis, Mo.). QIAprep Spin Miniprep, QIAGEN PlasmidPlus Midi, QIAquick Gel Extraction, Phusion DNA polymerase, and Ni-NTAresin were purchased from Qiagen (Seoul, Korea). Restriction enzymes andT4 ligase were purchased from New England Biolabs (Ipswich, Mass.).Alexa-labeled secondary antibodies, bacterial cell line DH10B, mammaliancell line HEK293-H, neuronal cell culture media, SAV conjugated withhorseradish peroxidase (SAV-HRP), Opti-MEM® I Reduced Serum Medium,Biocytin-Alexa 594, D-biotin, and Dynabeads® M-280 Streptavidin werepurchased from Invitrogen (Carlsbad, Calif.). Silica beads (5 μm indiameter) were purchased from Bangs Laboratories, Inc. (Fishers, Ind.).Egg phosphatidylcholine (PC) and1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(cap-biotinyl)(sodium salt) (Biotin-Cap-PE) were purchased from Avanti Polar Lipids(Alabaster, Al). Goat Nlg-1 polyclonal antibody (sc-14084) was purchasedfrom Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.). Rabbitsynapsin I monoclonal antibody (AB1543), guinea pig VGLUT1 polyclonalantibody (AB5905), and rabbit GAD65/67 antibody (AB1511) were purchasedfrom Merck Millipore (Billerica, Mass.). Mouse Bassoon monoclonalantibody (ab82958) was purchased from Abcam (Cambridge, UK).

Example 2 Molecular Biology

The predicted O-glycosylation motif (OG), transmembrane domain (TMD),and cytosolic domain of Nlg-1, from Ser640 to the C-terminus, werereplaced with a GS-linker followed by a 14-mer biotin acceptor peptide(AP or AviTag). First, pNICE-YFP-Nlg-1 and the staggered PCR productfrom primers AP-1F (SEQ ID NO: 2), AP-2R (SEQ ID NO: 3), AP-3F (SEQ IDNO: 4), and AP-4R (SEQ ID NO: 5) were digested with KpnI and NotI, andligated together. To aid purification, His×8 encoding primers, PvuI-H8-F(SEQ ID NO: 7), and PvuI-H8-R (SEQ ID NO: 8) were annealed, andintroduced upstream of the YFP sequence using a single PvuI site,yielding pNHY-Nlg-1-AP. To replace YFP with monomeric RFP, TagRFP-T wasPCR amplified from pcDNA3-TagRFP-T using primers PvuI-H8-TagRFP-T-F (SEQID NO: 9) and TagRFP-T-SalI-R (SEQ ID NO: 10), cut with PvuI and SalI,and ligated with pNHY-Nlg-1-AP that had been digested with the samerestriction enzymes, resulting in pNHR-Nlg-1-AP. To introduce RFP afterNlg-1, new cloning sites, PvuI and SalI, were inserted within the GSlinker using primers AP-1F, AP-2R, AP-PvuI-SalI-3F (SEQ ID NO: 6), andAP-4R. The staggered PCR product was then ligated withpNICE-HA-H6-Nlg-1ab-GPI after restriction digest of both DNAs with KpnIand NotI, yielding pNHH-Nlg-1-AP. The TagRFP-T PCR product (see above)and pNHH-Nlg-1-AP were digested with PvuI and SalI followed by ligationto give pNHH-Nlg-1-R-AP. Bacterial expression and purification ofbacterial BirA biotin ligase were conducted using pET21a-BirA plasmid.The primers used in the present invention are listed in Table 2 below.

TABLE 2 DNA oligomer sequence from Primer name 5′ end (length/bp)SEQ ID NO AP-1F GGCGGTGGTACCTCATCTGCATAATCTCAATGACATT  2GGCGGCGGCAGCGGCGGAGGCAGCGAGGG (66) AP-2RGCCCTCGCTGCCGCCTCCGCTGCCGCCTCCGCTGCCT  3 CCGCCCTCGCTGCCTCCGCCGCT (60)AP-3F GCGGAGGCGGCAGCGAGGGCGGAGGCAGCGGCGGCGG  4CCTGAACGACATCTTCGAGGCCC (60) AP-4R GGCAGCGCGGCCGCTTACTCGTGCCACTCGATCTTCT 5 GGGCCTCGAAGATGTCGTTC (57) AP-PvuI-SalI-GCGGAGGCGGCAGCGAGGGCCGATCGGGTGTCGACGG  6 3F CCTGAACGACATCTTCGAGGCCC (60)H8-PvuI-F CGCACCATCACCACCACCACCATCACCGAT (30)  7 H8-PvuI-RCGGTGATGGTGGTGGTGGTGATGGTGCGAT (30)  8 H8-TagRFP-T-GGCCGATCGCACCATCACCACCACCACCATCACATGG  9 PvuI-F TGTCTAAGGGCGAAGAG (54)H8-TagRFP-T- GCCACCGTCGACCTTGTCGTCGTCGTCCTTGTACAGC 10 SalI-RTCGTCCATGC (47)

FIGS. 1a and 1b schematically show the 3-dimensional structure of Nlg-1dimer, locations of major amino acids, and the constitution of domainsof the complex according to the present invention and those of variouscomplexes designed for comparison.

Example 3 Establishment of Stable Cell Lines

To the 1.5 mL of Opti-MEM I Reduced Serum Media was added 24 μg of eachNlg-1-encoding plasmid DNA (pNHY-Nlg-1-AP, pNHR-Nlg-1-AP, andpNH-Nlg-1-R-AP). Likewise, 60 μL of a 1.0 mg/mL PEI solution was addedto the 1.5 mL of an Opti-MEM solution. After incubation at 25° C. for 5minutes, the two solutions were mixed at room temperature for 30 minutesand added to HEK293-H cells grown to about 20% confluence in a culturedish with a diameter of 10 cm at 37° C. DMEM medium was replaced after 4hours of incubation. After three days, the cells were treated with G418at a final concentration of 800 μg/mL. The G418 treatment was repeatedwith a fresh medium after two days. After two weeks, single colonieswith brightest fluorescence signals were picked and seeded on a 24-wellplate. Among them, the best fluorescent colonies were repeatedlyselected until only one colony was left and the final best fluorescentcolony was seeded on a culture dish with a diameter of 10 cm for thesubsequent passage. The thus-established stable cell line was kept in aDMEM medium containing 100 μg/mL of G418.

Example 4 Preparation of Biotinylated Nlg-1 (Biotinylated Nlg-1)

The established HEK-293-H stable cell lines of expressing Nlg-1 weretransfected with pDisplay-BirA-ER plasmid. Specifically, the plasmidpDisplay-BirA-ER (24 μg) dissolved in 1.5 mL of the Opti-MEM solutionwas mixed with 1.56 mL of an Opti-MEM solution containing 60 μg of PEIat 25° C. for 20 minutes. The mixture was added to the establishedHEK293-H stable cell lines at about 20% confluence in a culture dishwith a diameter of 10 cm. After 4 hours of incubation, DMEM medium wasreplaced with a fresh one containing 100 μg/mL G418 and 10 μM biotin.The cells transfected with the plasmid were cultured for 3 days to 6days at 37° C. to allow the in vivo biotinylated Nlg-1 to be secretedinto the culture medium. Then, 10 mL of the medium was saved and thewhole cells were transferred to a culture dish with a diameter of 15 cmand filled with 30 mL of DMEM containing 100 μg/mL G418 and 10 μMbiotin. After another three days, the culture medium was combined withthe saved medium and subjected to column purification using 2 mL ofNi-NTA resin according to the manufacturer's protocol.

Meanwhile, for in vitro biotinylation, the stable cell line was grownwithout BirA transfection. Instead, the purified Nlg-1 was treated withBirA enzyme. Specifically, to 1 mL of a column elution fraction showingthe highest fluorescence signal was added 5 mM MgCl₂, 1 mM ATP, 0.1 mMbiotin, and 30 nM BirA enzyme as final concentrations and incubated at37° C. while shaking for 2 hours. The levels of in vivo and in vitrobiotinylation were analyzed via western blot using SAV-HRP or using goator mouse anti-Nlg-1 antibody and HRP-conjugated secondary antibody. Thepurity of Nlg-1 was analyzed by SDS-PAGE with silver staining, followedby quantification using NIH ImageJ software. The Nlg-1 concentrationobtained from the image analysis was compared to fluorescence intensitymeasured with a Synergy Mx fluorescence microplate reader (BioTek,Seoul, Korea).

Example 5 Reconstitution of Nlg-1-RFP-AP on Microbeads

Egg PC mixed with Biotin-Cap-PE (L-α-phosphatidylcholine, 99 mol % eggPC and 1 mol % Biotin-Cap-PE) in chloroform was dried, hydrated, andfilter-sterilized using PBS (1 mL, 100 mM, pH 7.4) to yield 5 mg/mLinitial concentrations of phospholipids. The SUV was generated byextrusion through 50 nm pores and diluted in PBS to a finalconcentration of 1 mg/mL. The solution (450 μL) was mixed with 1 μL ofautoclaved silica microbeads (about 3.0×10⁵ beads) at 25° C. for 30minutes and incubated while vortexing intermittently. After rinsingtwice with 1 mL PBS, the beads were incubated with PBS (1 mL) containingBSA (100 μg/mL) at 25° C. for 45 minutes. The beads were washed with PBS(1 mL), treated with SAV (170 nM for 1% (mol/mol) Biotin-Cap-PE), andincubated at 25° C. for 45 minutes. After rinsing three times with 1 mLPBS, the beads were treated with 1 mL of the biotinylated Nlg-1 solutionand incubated overnight at 4° C. For the display of biotinylated Nlg-1on polymeric microbeads, Dynabeads M-280 stock solution (1 μL, 6×10⁵ to7.0×10⁵ beads) was added to the Nlg-1 solution (1 mL) and incubated at25° C. for 3 hours. The thus-prepared Nlg-1 coated beads, SLB beads, andDynabeads were added to cultured hippocampal neurons (17 DIV) andincubated at 37° C. and 5% CO₂ atmosphere for 24 hours.

Example 6 Neuronal Cell Culture

Primary hippocampal neurons were obtained from Sprague-Dawley ratembryos at day 18 of gestation (E18). Specifically, hippocampi dissectedfrom E18 rat embryos were rinsed with HBSS, and then incubated withpapain and DNase at 37° C. while stirring at a rate of 60 rpm for 30minutes. After sequential rinsing with solutions of 10% and 5% FBS inHBSS, individual single cells were mechanically isolated by performingtrituration 10 times in 2 mL HBSS containing DNase with a silanizedPasteur pipette (the pipette tip was barely polished with fire). Thecell suspension was diluted to a density of 2×10⁵ cells/mL with aplating medium containing MEM supplemented with 0.6% (w/v) glucose, 10mM sodium pyruvate, 1 mg/mL FBS, and 1% penicillin-streptomycin. Then,the cell-medium solution plated on the PDK-coated glass was placed in aPetri dish. Three hours thereafter, the cell culture medium wasexchanged with a B27-supplemented neurobasal medium containing 2 mMglutamax. Cultures were maintained in an incubator at 37° C. and 5% CO₂atmosphere.

Example 7 Immunocytochemistry

Cells were fixed using 4% formaldehyde for 25 minutes and rinsed 3 timeswith PBS (100 mM, pH 7.4). The cells were then incubated in a blockingsolution, containing 4% BSA and 0.1% Triton X-100 dissolved in PBS, for30 minutes, and incubated in primary antibodies, diluted inTris-buffered saline (TBS, pH 7.4) containing 0.5% BSA and 0.1% TritonX-100, at 4° C. overnight. The samples were then washed three times withTBS and the fluorescent secondary antibodies were applied in TBScontaining 0.5% BSA solution at room temperature for 1 hour. The sampleswere washed again three times with TBS and once with DDW, and stored inVECTASHIED Mounting Medium containing DAPI at −80° C. until microscopicexamination. Fluorescence images were taken with a Zeiss LSM710 confocallaser scanning microscope equipped with ZEN 2009 software at theNational Center for Inter-university Research Facilities (NCIRF) ofSeoul National University (Korea).

Example 8 Image Quantification and Analysis

Fluorescence quantification was performed using NIH ImageJ software.Fluorescence intensities were measured from at least 10 beads under thesame experimental conditions and the data from at least three separateimmunostaining experiments was averaged. A fluorescence ratio wasdetermined by measuring the intensity of each channel of the same regionof interest (ROI) that includes augmented signals around the beads.

Experimental Example 1 Preparation and Purification of Proteins

The present inventors attempted to confirm the functional interactionsbetween the complex including a fluorescent protein, biotin, and Nlg-1and cultured hippocampal neurons, independent of SLB media. Thefluorescence tag can aid in establishing stable cell lines, and thusmass production, quantification, and tracking of Nlg-1 on a givenartificial substrate. Nlg-1 conjugated to theglycosylphosphatidylinositol (GPI)-anchoring motif is known to maintainits activity both in vivo and in vitro. As such, a complex in which aGPI-anchoring motif was conjugated at its C-terminus was prepared andused as a comparative example.

In the present invention, to facilitate protein purification, solubleand secreted forms of Nlg-1 were used. Although the Nlg-1-GPI containsLeu48-Pro631 of extracellular globular region, the crystal structure ofNlg-1/Nrx-10 complex revealed that Leu636, as the end of the α-helix,was required for Nlg-1 dimerization (FIG. 1a ). Additionally, Nlg-1-638was the minimum domain functionally secreted to a culture medium,whereas Nlg-1-626 and Nlg-1-633 were not. Since the GPI motif linked toNlg-1-631 begins with a KLLSATA amino acid sequence that has a highα-helical propensity, the overall Nlg-1-GPI structure may have remainedunaltered. Accordingly, the present inventors retained Nlg-1-639, whichincludes the minimum domain functionally secreted, and replacedO-glycosylation-rich domain (OG)-transmembrane domain (TMD)-cytoplasmicdomain (CD) domains with glycine-serine (GS) linker and a biotinacceptor peptide (AP) tag to maintain the functional structure of Nlg-1(FIG. 1b ). The AP-tagged Nlg-1 was biotinylated in vivo by transienttransfection of the Nlg-1 expressing stable cell lines with endoplasmicreticulum (ER)-specific BirA plasmid (FIG. 2). The quantification of thethus-prepared proteins was quantified using analytical methods such aselectrophoresis and western blot, based on the BSA prepared at a knownconcentration (FIG. 3).

Experimental Example 2 Reconstitution of a Complex on a Substrate, andEffects According to the Kinds of Fluorescent Proteins within a Complexand their Positions

A biotinylated Nlg-1 complex including a fluorescent protein accordingto the present invention was conjugated on a substrate. As thesubstrate, silica microbeads coated with phospholipids containing alipid biotin tag (BTN-SLB Beads) and streptavidin-coated Dynabeads(Dynabeads SAV) without a lipid bilayer were used. The lipid membranewas used so that the biotin-tagged lipid was contained in an amount of1%, and in particular, about 9×10⁵ complexes were conjugated per beadwith a diameter of 5 μm. The amount of the complexes conjugated to eachbead can be increased or reduced by adjusting the ratio of biotin-taggedlipid within the total phospholipids. For example, the present inventorshave confirmed that a stronger fluorescent signal appeared when thecomplex was conjugated to the beads containing the biotin-tagged lipidin an amount of 10%. This result is contrasted by the previous reportthat 80 to 480 Nlg-1-GPI proteins per 5 μm diameter silica bead arerequired for neuronal activation, and from the fact that a higher numberof complexes can be conjugated per unit bead, it was confirmed that thelifetime of the artificial synapse inducers conjugated to substrates forneuronal activation can be extended. In the case of Dynabeads on whichthe number of active sites of SAV molecules was optimized, thefluorescence signal was stronger than that of the SLB-silica beads (FIG.4, left vs. right), and in particular, the binding affinities wereconfirmed by comparing the fluorescence intensity of fluorescentproteins by respectively conjugating the complexes, which were preparedby varying the kinds of the fluorescent proteins and the positions ofthe fluorescent proteins and Nlg-1, to BTN-SLB beads and Dynabeads SAV.As a result, as shown in FIG. 3, the fluorescence intensity wasincreased more in the complex containing YFP as a fluorescent proteinthan in the complex containing RFP as a fluorescent protein (FIG. 4, YNvs. RN). In particular, it was confirmed that when the fluorescentprotein was conjugated at the C-terminus of Nlg-1 in the sequence ofNlg-1-RFP-biotin from the N-terminus, the fluorescence intensity wassignificantly increased (FIG. 4, RN vs. NR). This can be explained bythe fact that YFP itself can be dimerized to thereby inhibit thedimerization of Nlg-1, and that YFP can be more easily photobleachedthan RFP.

Additionally, the effect of the kinds of fluorescent proteins on theNlg-1 activity was confirmed. Although N-terminally YFP-tagged Nlg-1 wasproven to have a functional role in synaptogenesis, in vivo biotinylatedYFP-Nlg-1-AP was rarely functional on both SLB membrane beads and oncommercially available Dynabeads M-280 coated with an optimum amount ofSAV (FIGS. 5a and 5b ). In the present invention, in order to avoidpotential hindrance originating from YFP dimerization andphotobleaching, YFP was replaced with TagRFP-T16, a photostablemonomeric RFP, to thereby yield Nlg-1 with improved activity (FIGS. 5cand 5d ). Consequently, only the Nlg-1-RFP-AP complex, in which Nlg-1was conjugated to the N-terminal direction of TagRFP-T, was able toinduce presynaptic differentiation, recruiting synapsin I and vesicularglutamate transporter 1, which are presynaptic marker proteins (VGlut1)(FIGS. 6a and 6b ).

Experimental Example 3 Selective Induction of an Excitatory PresynapticDifferentiation

Presynaptic differentiation has been conventionally induced by polybasicmaterials, such as poly-D-lysine (PDK) and phosphatidylethanolamine(PE). In this regard, the present inventors used PDK microbeads ascomparative example. They confirmed that presynaptic differentiation canbe induced in neurites in contact with PDK microbeads by introducingboth synapsin I and VGlut1, as previously reported (FIG. 6c ). However,the expression levels of aggregated presynaptic markers were differentfrom each other. Specifically, synapsin I, a synaptic marker, gatheredaround the beads of both PDK and Nlg1-RFP-AP beads with similarintensities (FIGS. 6a to 6c and FIGS. 5 to 10). In contrast, theaggregation level of VGlut1, a representative excitatory presynapticmarker, was significantly low in the case of PDK beads compared toNlg-1-RFP-AP beads (FIG. 6d ). Additionally, the neurite-contactingNlg-1-RFP-AP beads showed a significantly higher rate of synapsin Iaggregation than the neurite-contacting PDK beads (FIG. 9). Theseresults suggest that Nlg-1-RFP-AP is a more potent inducer forexcitatory glutamatergic synapses than PDK for cultured hippocampalneurons.

Meanwhile, synapsin I is widely used as a general synaptic marker, butits distribution in neurons is quite delocalized such that the synapsinI puncta are frequently observed in the absence of synapses. Therefore,it is necessary to confirm the relationship between synapsin I punctaand synapses using other presynaptic markers. The present inventorsconfirmed, in addition to synapsin I and VGlut1, the expression ofBassoon protein capable of labeling the presynaptic active zone, whichis the site for secretion of neurotransmitters and is the nearest sitedirectly paralleled with the postsynaptic density (PSD). In thehippocampal neurons, cytomatrix protein Bassoon and the synaptic vesicleprotein synapsin I showed different distribution profiles. Bassoonmainly resides within about 70 nm from the synaptic cleft, whereassynapsin I populates within the region of about 70 nm to 200 nm distantfrom the synaptic cleft. In the present invention, given the diffractionresolution limit of confocal microscopy is about 200 nm to about 250 nm,it was confirmed that the enhanced synapsin I puncta were alwaysaccompanied by and were distinguished from the enhanced Bassoon puncta,with Bassoon being closer to the beads (FIGS. 7a to 7c , and FIG. 11).Additionally, the neurite-contacting Nlg-1-RFP-AP beads showed asignificantly higher rate of Bassoon aggregation than theneurite-contacting PDK beads (FIG. 11).

Additionally, the present inventors confirmed the specificity ofNlg-1-RFP-AP to excitatory presynaptic differentiation by comparing withthe expression of glutamic acid decarboxylase (GAD), a presynapticmarker protein for the inhibitory GABA synapses. Not only theNlg-1-RFP-AP complex on the SLB beads but also that on the non-SLB beadsshowed higher preference for the excitatory presynaptic marker, VGlut1,but not for GAD (FIGS. 8a to 8d , and FIG. 12). The GAD, in general,showed discrete strong background signals with random distribution andin an all-or-none fashion, yielding stochastic colocalization withVGlut1, which were ruled out in fluorescence intensity calculations(FIG. 12c , arrow). As in FIG. 6c , the PDK beads induced an increase inthe expression level of VGlut1, but there was also an increase of GADpopulation around the PDK beads, as previously reported (FIGS. 8c and 8d). Additionally, there were occasional GAD-positive only puncta (FIG.12d , arrow) as well as VGlut1- and GAD-positive puncta (FIG. 12d ,arrowhead). Overall, the PDK beads gave bead-shaped GAD-positive puncta,whereas the Nlg-1 beads did not.

Lastly, the present inventors confirmed that the signals of synapticmarkers increased as the fluorescence intensity from the Nlg-1-RFP-AP,which was conjugated to a substrate, increased, i.e., as the number ofthe complexes increased, regardless of the kinds of the substrate. Thisindicates that the activity of Nlg-1-RFP-AP is irrelevant to the methodof immobilizing it to a substrate, and it simply relies on the degree ofconjugation.

The mechanism of inducing an excitatory presynaptic differentiation by acomplex, according to the present invention, containing a polypeptide,which includes an extracellular domain of Nlg-1, RFP, and biotin taggedat the C-terminus in this order is shown in FIG. 13. Additionally, theprinciple of inhibition of presynaptic differentiation in a complexcontaining RFP in the N-terminus is also illustrated therein along witha non-specific differentiation mechanism by a polybasic substrate suchas PDK.

Experimental Example 4 Difference Between Synapses Induced by Nlg-1 andPDK

In order to confirm the difference between the synapses induced byNlg-1, a synaptogenic protein, according to the present invention, andthe synapses induced by PDK, a polybasic material which has beenconventionally used for induction of presynaptic differentiation, thebeads coated with Nlg-1 beads and PDK, respectively, i.e., Nlg-1 beadsand PDK beads, were cultured after addition thereof to neurons, and theinduced synapses were compared by immunochemical fluorescence analysis.In order to confirm the difference according to culture period andcontacting hours, experiments were performed by adding beads atdifferent points of culture. The cells isolated in Example 6 were usedas the neurons, and the neurobasal medium described in Example 6 wasused and cultured. The result of the immunochemical fluorescenceanalysis is shown in FIG. 14.

As shown in FIG. 14, when the cells were cultured with the beads, thePDK was recognized as a simple adhesion protein, and thus the inductionof a synapse failed (FIG. 14a ). This is also supported in theliterature by Dr. Colman (A. L. Lucido et al., 2009,29(40):12449-12466). Meanwhile, it was confirmed that the Nlg-1 beadsinduced a concrete formation of synapses if in contact with cellsregardless of the developmental stage of the cells added thereto (0 DIVto 17 DIV) (FIGS. 14a to 14e ).

Additionally, the synapses formed by the Nlg-1 beads were firmlymaintained as the culture hours increased, whereas the synapses formedby PDK were either weakened or lost without long-term maintenance (FIGS.14a to 14e ).

From the above results, the synapses formed by Nlg-1, a synaptogenicprotein, according to the present invention, were shown to havecharacteristics different from those of the synapses formed by PDK, apolybasic material. Accordingly, considering that the formation ofsynapses which are firm and can be maintained long-term are required forthe establishment of a new neural network through artificial synapses,an artificial synapse inducer including a synaptogenic protein such asNlg-1, which can form a synapse by contacting with neural cellsregardless of the developmental stage of the cells and maintain theformed synapse long-term, is preferable for the purpose of forming aneural interface capable of simulating the real brain environment.

1. A complex comprising a polypeptide comprising an extracellular domainof a synaptogenic protein; and biotin tag at the C-terminus of thepolypeptide.
 2. The complex of claim 1, wherein the synaptogenic proteinis selected from the group consisting of neuroligin, neurexin,leucine-rich repeat transmembrane protein (LRRTM), netrin G ligand(NGL), synaptic cell adhesion molecule (SynCAM), ephrin-B receptor(EphB), leukocyte common antigen-related protein (LAR), netrin G, andSlitrk (Slit- and Trk-like proteins).
 3. The complex of claim 1, whereinthe synaptogenic protein is neuroligin-1.
 4. The complex of claim 1,further comprising a fluorescent protein in the order of the polypeptidecomprising an extracellular domain of a synaptogenic protein, thefluorescent protein, and the biotin from a N-terminus thereof.
 5. Thecomplex of claim 4, wherein the fluorescent protein is red fluorescentprotein (RFP).
 6. The complex of claim 1, further comprising apolyhistidine-tag (His-tag) or influenza hemagglutinin epitope tag(HA-tag).
 7. An artificial synapse inducer, wherein the complex of claim1is attached to a substrate coated with a biotin-binding protein.
 8. Theartificial synapse inducer of claim 7, wherein the biotin-bindingprotein is selected from the group of avidin-like proteins consisting ofstreptavidin, traptavidin, and neutravidin.
 9. The artificial synapseinducer of claim 7, wherein the substrate is a microbead.
 10. A methodfor preparing a presynaptic differentiation-induced neuron comprisingculturing the neuron in a medium comprising the artificial synapseinducer of claim
 7. 11. The method of claim 10, wherein the presynapticdifferentiation is an excitatory presynaptic differentiation or aninhibitory presynaptic differentiation.
 12. The artificial synapseinducer of claim 7, wherein the synaptogenic protein is selected fromthe group consisting of neuroligin, neurexin, leucine-rich repeattransmembrane protein (LRRTM), netrin G ligand (NGL), synaptic celladhesion molecule (SynCAM), ephrin-B receptor (EphB), leukocyte commonantigen-related protein (LAR), netrin G, and Slitrk (Slit- and Trk-likeproteins).
 13. The artificial synapse inducer of claim 7, wherein thesynaptogenic protein is neuroligin-1.
 14. The artificial synapse inducerof claim 7, wherein the complex further comprises a fluorescent proteinin the order of the polypeptide comprising an extracellular domain of asynaptogenic protein, the fluorescent protein, and the biotin from aN-terminus thereof.
 15. The artificial synapse inducer of claim 14,wherein the fluorescent protein is red fluorescent protein (RFP). 16.The artificial synapse inducer of claim 7, wherein the complex furthercomprises a polyhistidine-tag (His-tag) or influenza hemagglutininepitope tag (HA-tag).